Of the two V. cholerae chromosomes, the larger one (Chr1) carries most of the housekeeping genes and is considered the primary chromosome. The smaller chromosome (Chr2) seems to have evolved from a plasmid. Plasmids, although prevalent in bacteria, rarely serve as the source of replicons that drive chromosomal replication. One reason could be that the firing of plasmid origins is generally not restricted to a specific time of the cell cycle, whereas timely firing is the norm for chromosomal origins in all domains of life. Comparison of plasmid and Chr2 systems could thus be valuable to understand how the timing of a biological process is regulated in the cell cycle. Chr2 replication seems also to depend on ChrI replication. How the two chromosomes communicate to coordinate replication is largely unknown and this knowledge is basic to our understanding of how multipartite genomes are maintained in bacteria. In eukaryotes, in addition to incomplete and over-replication, uncoordinated replication causes developmental abnormalities and cancer. Finally, Chr2 replication is controlled by a segregation protein. The discovery of the influence of segregation on replication is a recent development in the field of chromosome dynamics in bacteria, and cross-talk between replication and segregation has been demonstrated recently in yeast and human cells. Our progress in understanding these processes is reported below. Transition from random to cell-cycle regulated replication initiation: The initial characterization of Chr2 replication suggested that its control would be more complex than that of its presumed progenitor plasmid. Our past studies identified three new features of the chr2 origin: 1) Requirement for methylation of initiator binding sites in the origin for initiator binding. Several bacterial origins have methylation sites and timely firing of origins depends on methylation; 2) Presence of a new kind of initiator binding site (39-mers) in the origin exclusively for inhibiting over-replication; and 3) Presence of an extra regulatory feedback loop to control initiator synthesis more tightly. In 2013-2014, we discovered three additional regulators in the control of chr2 replication, which we are continuing to characterize. The cell-cycle regulation of replication initiation thus appears to be considerably more involved than the mechanisms that regulate plasmid replication. The mechanisms of action of the new regulators are discussed below. 1. Participation of initiator dimers in the control of chr2 replication: The chr2 initiator does not have any enzymatic activity but uses homodimerization to regulate its activity. Only monomer binding to the replication origin leads to initiation. The dimers compete with monomers for binding to the origin and thus hinders initiation. The dimerization also precludes initiator binding to replication inhibitory 39-mer, which binds only the monomers. Controlling the association state of the initiator is thus critical for controlling Chr2 replication. We have determined the domains of the initiator involved in origin and 39-mer DNA binding and in dimerization. Important residues for all three activities are found to reside within 71 out of the 658 residue long protein. The domains for the three activities also overlap explaining how changes in one domain can influence the activities of the other two domains. The interplay between dimerization and DNA binding is well-known in gene regulation but it seems to play a more profound role in controlling Chr2 replication. 2. Control of Chr2 replication by a site in Chr1: We hypothesize that the timely replication and segregation of the two V. cholerae chromosomes would require communication between them, so that both the processes can complete prior to cell division. Preliminary evidence for inter-chromosomal communication has been obtained. We identified a locus on Chr1 that can significantly stimulate Chr2 replication, likely by remodeling theChr2 initiator. The DNA site also made the normally chaperone (DnaK)-dependent replication of Chr2 chaperone-independent. For many DNA binding proteins, specific binding is known to remodel the protein. The novelty of the Chr1 site is that its sequence has no similarity to the known specific binding sites of the initiator. Characterization of this atypical DNA-protein interaction is our goal for the immediate future. In any event, the finding suggests a mechanism that may coordinate replication of the two chromosomes. 3. Control of Chr2 replication by a segregation protein: The fact that segregation can influence replication was not known until recently. Our knowledge of chromosome segregation in bacteria comes primarily from studies of plasmids, where genes dedicated to segregation (par genes) were first found. Homologues of plasmid par genes have now been identified near the origin of replication in most bacteria, including V. cholerae. Both of the Vibrio chromosomes have their own par genes. We showed earlier that one of the par genes (parA1) of Chr1 specifically promoted Chr1 replication. A similar finding was also made in B. subtilis. In both B. subtilis and V. cholerae, the universal bacterial initiator, DnaA, was found to be the direct target of Par proteins. We now find that ParB2 of Chr2 can also specifically stimulate replication of Chr2 but through different mechanisms. ParB2 could spread beyond the centromere into the adjacent replication origin and cover one of the replication inhibitory 39-mer sites. ParB2 binding to the 39-mer is likely to interfere with initiator binding to the 39-mer, which is required for replication inhibition. ParB2 thus could help promote replication by interfering with the inhibitory activity of the 39-mer. Unexpectedly, ParB2 could also bind to another 39-mer directly without requiring spreading from a centromeric site. ParB2 thus appears to promote replication by tempering the activities of the two 39-mers, the two strongest replication inhibitory sites, by spreading into one and directly binding to the other. These studies establish several ways by which segregation proteins could influence replication. Generation of Vibrio-specific antimicrobial agents: Given the increasing prevalence of multi-drug resistant pathogenic vibrios, there is a need for new targets and drugs to combat these pathogens. The Chr2 initiator, RctB, is conserved only in the family Vibrionaceae and appears ideally suited for developing potential anti-vibrio specific drugs. 3-D structural information of target proteins greatly facilitates drug design. Towards this goal we have started a systematic domain analysis of RctB. Replication initiators in general have proven refractory to structural studies most likely because they have unstructured regions and require remodeling by chaperone proteins and/or binding to specific DNA for activity. In collaboration with Alex Wlodawer (CCR), although initial attempts to form crystals of RctB have failed, we are attempting deletion analysis to isolate functional domains, hoping that smaller functional regions might be better suited for crystallization. We are also trying to identify domains by partial proteolysis and identity the products by MassSpec (in collaboration with Lisa Jenkins, CCR). These smaller derivatives might be more amenable to structural studies. We are particularly optimistic that structure determination of the 71 amino acids long fragment described above will be possible by NMR (in collaboration with Yawen Bai, CCR) because of its small size. This small region could be an attractive drug target as it contains three important domains of the initiator.